The invention refers to the field of scanning microscopy, in particular to the field of confocal scanning microscopy. Scanning microscopes have been known from practical use for years. Reference is made in this connection, merely by way of example, to DE 196 54 211 A1. For purposes of the present invention, scanning microscopes are furthermore all microscope types that comprise scanning image detection or image construction. Control computers for such scanning microscopes are usually personal computers that have special interfaces for controlling the microscope hardware. Control computers for purposes of the present invention can also be single-board computers, special computer hardware integrated into the scanning microscope, or an entire computer cluster. Computer clusters serve not only to control the scanning microscope but also to perform farther-reaching calculation-intensive operations, for example the visualization of multidimensional image data. The image data acquired using a confocal scanning microscope usually encompass two-dimensional image data—so-called “optical sections” of a specimen. Depending on the task, three-dimensional image data sets or one-dimensional line scans are also performed, the specimen being confocally illuminated with light of one or more light sources, possibly of different wavelengths. In the case of fluorescence microscopy, single- or multiple-photon processes for exciting the fluorescent markers or specimens are usual.
Confocal scanning microscopes in particular require that the user have sufficient knowledge about the operation of such a scanning microscope, specifically in order to set the mutually dependent system parameters that often also work against one another or are mutually exclusive. These include the pinhole diameter of the confocal detector unit, the high voltage of the photomultiplier (PMT) of the detector unit, the laser power level, etc. For optimum setting of the system parameters, especially in consideration of specimen-specific properties, the user must utilize his or her experience with using such scanning microscopes. Hitherto, however, it has been almost impossible for a user to achieve optimum imaging results without comprehensive relevant experience.
Because of the aforementioned complexity involved in setting confocal scanning microscopes, the operating or system parameters of scanning microscopes are not optimally set by many users. Lack of understanding of the often very complex correlations among various optical and electronic boundary parameters of a confocal scanning microscope is, in particular, the cause of hitherto inadequate operation. But if a scanning microscope of this kind is not optimally set, an image can be acquired only with reduced image quality or with far too long a setting procedure prior to the actual image acquisition. Too long a setting phase prior to the actual image acquisition reduces the efficiency of such a microscope, however, and usually results in excessive wear on the laser light source and/or the light-guiding fibers acted upon by the laser light, and possibly in negative effects on the specimen.
The scanning microscopes hitherto known from practical use are also problematic, in particular, in terms of training new users, since instruction and assistance from experienced users is always necessary. It has hitherto been difficult, however, to learn to use a scanning microscope optimally on a self-taught basis. To the contrary, with existing scanning microscopes an extremely long training phase with the assistance of experienced users is absolutely necessary.
A further problem from existing practice is that many fluorescent specimens become bleached out in the course of very long setting periods. Since long setting periods cannot, however, usually be ruled out, the use of scanning microscopes hitherto known from practical use is limited, especially in the context of biological specimens, and thus problematic. There is thus a need for a reduction in the time required for optimum setting.